Popping Out MX Cassettes from Saccharomyces cerevisiae

Abstract

MX cassettes are frequently used to generate knockout (KO) mutations in Saccharomyces cerevisiae. The recycling or “popping out” of an MX cassette flanked by direct repeats allows the same cassette to be reused in a strain to generate additional KO mutations. Popping out MX cassettes also eliminates MX homology in a strain, which facilitates the subsequent generation of additional KO mutations with other MX cassettes. MX cassettes can be recycled or “popped out” of the genome by spontaneous recombination between large, cassette-borne MX3 or PR direct repeats and by Cre-mediated, site-specific recombination between small, cassette-borne loxP direct repeats. Both of these techniques leave a mutation with a cassette-encoded “scar.” For the URA3MX, LYS5MX, FCA1/FCY1MX, and amdSYM cassettes, there are counterselections. Counterselections are extremely useful as they allow for positive selection for plasmid shuffling, transplacement of mutant alleles into the genome, and recycling or popping out cassettes flanked by cassette-encoded direct repeats to yield mutations with a cassette-encoded scar. Finally, after amplifying with the appropriately designed primers, integrated counterselectable MX cassettes can be popped out to generate seamless or “scar-free” deletion mutations, as well as indel and point mutations.